MECHANISM OF INOSITOL TRISPHOSPHATE ACTION

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DESCRIPTION: An elevation of the free calcium concentration in the cytoplasmic compartment is an integral component of the mechanism by which cells respond to hormones, growth-factors and certain neurotransmitters. D- myo-Inositol 1,4,5-trisphosphate (IP3) is an intracellular messenger mediating the hormonal mobilization of Ca2+ from intracellular stores. This molecule interacts with a specific receptor (IP3R) that has been purified and shown to be a ligand-gated Ca2+ channel. The central theme of this proposal is to study the structure, function and regulation of IP3 receptors. All the studies proposed utilize recombinant receptor fragments or cerebellum membranes, hepatocytes or WB-cells (a rat liver epithelial cell line). The specific aims of the proposal are: 1) Determination of the topology of the transmembrane domains and mechanism of their membrane insertion. This will be studied using a cell-free translation/translocation assay programmed with cRNA encoding the putative transmembrane domains. The system will be used to experimentally test controversial models of transmembrane organization of the receptor based on hydropathy analysis and to identify transmembrane domains important for homo- and heteroligomerization; 2) Study the mechanism of IP3R regulation by Ca2+ and phosphorylation. Recombinant fusion proteins and proteolytically cleaved domains of the receptor will be used to identify Ca2+ binding sites on the receptor and interactions with Ca2+ regulatory proteins. The protein kinases and phosphatases that regulate IP3R function will be identified. The basis for the differential regulation of neuronal and peripheral IP3R isoforms by Ca2+ and phosphorylation will be investigated. 3) To study interaction of the receptor with the cytoskeleton. Interaction of the Type-I IP3R with ankyrin will be further analyzed and the interaction of the WB-IP3R with the cytoskeletal matrix will be characterized. 4) To study interactions between different domains of the receptor and between different receptor isoforms. Recombinant fusion proteins and proteolytically cleaved domains of the receptor will be used to further localize the ligand- binding domain and to study its interaction with the C-terminal channel domain. Initial observations on heteroligomerization of type-I and type- III IP3R will be further investigated. This proposal is focused on obtaining basic information on IP3R proteins. The long-term goal is to understand how these proteins function in individual cells to generate complex spatial and temporal patterns in their Ca2+ transients and how such signals are decoded to alter physiological responses.